Human red blood cells in vivo are in a dynamic state. In whole blood, white blood cells are normally present in the range between 4,300 and 10,800 cells/μL and the normal RBC range at sea level is 5.4 million/μL (±0.8) for men and 4.8 million μL (±0.6) for women. The red blood cells contain hemoglobin, the iron-containing protein that carries oxygen throughout the body and gives red blood its color. The percentage of blood volume composed of red blood cells is called the hematocrit.
The normal life span of a red blood cell (RBC) is 120 days. Approximately 0.875% of the RBCs are retired every 24 hours by the spleen and new RBCs are made by the bone marrow. Consequently, when blood is drawn from a donor, there are a percentage of white blood cells and a spectrum of cells of different ages.
During the 6 weeks of conventional cold storage, there is a time-dependent deterioration of the biochemical, molecular, and mechanical properties of the RBCs collectively known as the “RBC storage lesion.” Upon collection, whole blood is introduced to citrate phosphate dextrose (CPD), an acidic solution, which immediately lowers the blood pH to 7.1 from a normal pH of 7.4 and leads to the rapid breakdown of 2,3-diphosphoglycerate (2,3-DPG) within the RBC; by 2 weeks of storage, 2-3-DPG is undetectable. At 1-6° C., the metabolic processes of RBCs are not entirely halted. RBCs, suspended in dextrose, metabolize dextrose into adenosine 5′-triphosphate (ATP), 2,3-DPG and metabolic waste products, such as lactate. Reactive oxygen species (e.g. hydroxyl, peroxy, alkoxy radicals) are generated by the inability of the refrigerated RBC, which contains a highly reactive mixture of iron and oxygen, to maintain the iron atoms of hemoglobin (Hb) in a ferrous state.
The continual exposure of the RBCs to accumulated waste products and reactive oxygen species leads to a decrease in RBC deformability via an increase in membrane rigidity, a decrease in the surface area/volume ratio, and increase in intracellular viscosity. Progressive oxidative damage to the proteins, lipids and carbohydrates of the RBC membrane results in increased ion leaks (e.g., potassium) and loss of membrane by exocytosis (resulting in a gradual change in the morphology and size of the RBCs from 8 μm diameter biconcave disks to 5 μm diameter spherocytes), increased mean cell hemoglobin concentration, increased exposure of phosphatidylserine (PS, a known marker for RBC senescence and clearance from circulation) on the outer surface of the membrane—PS increases RBC adhesiveness to the endothelium and procoagulant activity—and increased osmotic fragility. These effects ultimately result in RBC apoptosis, hemolysis, and the accumulation of extracellular free hemoglobin in the bag. Oxidative damage to the RBCs increases the acidity and hyper-osmolality of the suspending storage media (with an end pH of ˜6.6 and osmolality of ˜320 mmol/kg at 6 weeks); the deteriorating RBCs are bathed in an increasingly toxic media. When transfused, intravascular hemolysis may occur, as well as acute hypertension, vascular injury, kidney dysfunction and/or rapid clearance from the circulation when transfused.
The RBC expends ATP in an attempt to repair its cellular components; however, without a nucleus, the protein synthesis needed to continually replace key enzymes within the RBC cannot occur throughout the duration of storage, so the cells become increasingly rigid and fragile. Storage induced biochemical and mechanical changes within the RBCs reduce their ability to undergo physiologically relevant deformations, increasing the overall viscosity of blood and decreasing microvascular perfusion (e.g., at bifurcations, non-deformable RBCs may bypass the narrow microchannels for the larger, high flow microchannels or even plug the narrow microchannels as seen in our devices) resulting in poor tissue oxygenation.
The primary physiologic function of red blood cells (RBCs) is to transport oxygen from the respiratory surfaces in the lungs to the metabolizing tissues and end organs. The level of local tissue oxygenation depends to a large degree on the dynamics of blood flow in microchannels of microvascular networks. To maintain adequate microvascular perfusion, RBCs must be able to continually deform at physiologically high hematocrit (Hct) concentrations, under a wide range of flow conditions, in vessels ranging from 3- to 8-μm microchannels to 50- to 100-μm arterioles and venules. Therefore, maintaining an appropriate level of “deformability” is crucial for RBC physiological function. The RBC's ability to undergo folding deformations when traversing narrow microchannels and shear-induced deformations of the kind experienced by RBCs in larger vessels, arterioles, and venules declines progressively in storage. Because of this storage-induced deterioration, stored RBCs may be unable to maintain adequate blood flow in microchannels and deliver oxygen as effectively as their fresh counterparts. If transfused, they may be unable to improve perfusion of the microvascular networks and oxygenation of tissues and, thus, may reduce the clinical efficacy of RBC transfusions. The least deformable stored RBCs are mechanically sensed and retained by the spleen and removed from circulation by the reticuloendothelial system shortly after transfusion.
The decline of RBC deformability during refrigerated storage is a very well-known part of “storage lesion” and has been previously extensively documented in studies employing a wide variety of existing methods. The comparison between fresh and stored RBCs to test the overall sensitivity of a CND of the disclosure is used in the context of RBC storage and transfusion (rather than to demonstrate the progressive deterioration of RBC mechanical properties occurring in storage).
Most of the existing tools for measuring the deformability of RBCs have been applied to stored cells including: 1) micropipette aspiration, 2) micropore filtration, 3) ektacytometry, 4) laser-assisted optical rotational cell analyzer (LORCA), 5) optical tweezers, and 6) RBC adhesion assay. Although very informative for studying the RBC rheologic response under well-defined conditions, these conventional approaches have several common limitations: 1) each technology is focused on a narrow subset from the wide spectrum of deformations experienced by RBCs in the microcirculation, 2) RBC properties are examined under nonphysiological conditions (e.g., at ultralow Hct or while suspended in high-viscosity buffers), and 3) the effects of the architecture of microvascular networks are not taken into account.
The flow of blood in actual microvascular networks has been found to exhibit chaotic temporal oscillations (Griffith, “Temporal Chaos in the Microcirculation,” Elsevier, Cardiovascular Research, Vol. 31, p. 342-358, 1996). Kiani et al. proposed that such oscillations may occur in the absence of biological regulation and could result entirely from specific nonlinear rheological properties of blood within microvascular networks (Kiani et al., “Fluctuations in Microvascular Blood flow Parameters Caused by Hemodynamic Mechanisms,” The American Physiological Society, p. H1822-H1828, 1994). However, direct validation of microvascular behavior using in vivo experiments may be prohibitively complex or even impossible. Thus, an in vitro model of microvascular networks is essential for critical evaluation and/or validation of existing theory, models and computer simulations.
Cokelet et al. described a simple modification of standard photolithography that was used to fabricate systems of interconnected microchannels (Cokelet et al., “Fabrication of in Vitro Microvascular Blood Flow Systems by Photolithography,” Microvascular Research, Vol. 46, p. 394-400, 1993). This and other methods have been successfully adapted for production of flow systems that are used for endothelial cell culture (Frame et al., “A System for Culture of Endothelial Cells in 20-20-μm Branching Tubes,” Microcirculation, Vol. 2, No. 4, p. 377-385, 1995; US Pat. Pub. 2002/0182241, Borenstein et al., 2002; U.S. Pat. No. 7,517,453, Bitensky, et al., 2009).
However, current instrumentation does not provide sufficient and adequate means to obtain actual information regarding the flow of blood in capillary network channels. Accordingly, there is a need for improved artificial microvascular network devices to simulate the microvascular system. There is a further need to be able to assess the quality of stored blood prior to transfusion into patients. In addition, there is a need to assess the deformability of a blood sample prior to preparing the blood for long term storage. Finally, there is a need to be able to identify blood subjected to long term storage that has an increased risk of adverse events when transfused into a patient.